P53-based Cancer Therapy Research Articles

A structure-based virtual screening identifies a novel MDM2 antagonist in the activation of the p53 signaling and inhibition of tumor growth.

p53, a tumor suppressor protein, has a vital role in the regulation of the cell cycle, apoptosis, and DNA damage repair. The degradation of p53 is predominantly controlled by the murine double minute 2 (MDM2) protein, a ubiquitin E3 ligase. The overexpression or amplification of MDM2 is commonly observed in various human cancers bearing wild-type p53 alleles, leading to the rapid degradation of the p53 protein and the attenuation of p53 tumor suppression functions. Thus, a major effort in p53-based cancer therapy has been to research MDM2 antagonists that specifically stabilize and activate p53, leading to the suppression of tumor growth. However, despite numerous efforts to develop MDM2 antagonists, to date they have failed to reach clinical use, largely because of the cytotoxicity associated with these small molecules. This study used our newly designed structure-based virtual screening approach on a commercial compound library to identify a novel compound, CGMA-Q18, which directly binds to MDM2, leading to the activation of p53, the induction of apoptosis, and cell cycle arrest in cancer cells. Notably, CGMA-Q18 significantly inhibited tumor xenograft growth in nude mice without observable toxicity. These findings highlight our useful virtual screening protocol and CGMA-Q18 as a putative MDM2 antagonist.

Mdm2 Targeting Reassembly Peptide (Trap) Nanoparticles For P53 Based Cancer Therapy.

Gene mutations and functional inhibition are the major obstacles for p53-mediated oncotherapy. For p53-wild type tumors, the underlying mechanisms of functional inhibition of p53 during oncogenesis are unknown. Our results revealed that the expression of the MDM2 inhibitor ARF was inhibited in p53-wild type tumors, indicating that the restoration of ARF could be a potential oncotherapy strategy for p53-wild type tumors. Therefore, we developed ARF-mimetic MDM2-targeting reassembly peptide nanoparticles (MtrapNPs) for p53-based tumor therapy. Our results elucidated that the MtrapNPs responded to and formed a nanofiber structure with MDM2. By trapping MDM2, the MtrapNPs stabilized and activated p53 for the inhibition of p53-wild type tumors. In most cases, reactivated mutant p53 is inhibited and degraded by MDM2. In the present study, MtrapNPs were used to load and deliver arsenic trioxide, a p53 mutation rescuer, for p53-mutated tumor treatment in both orthotopic and metastatic models, and they exhibited significant therapeutic effects. Therefore, our study provides evidence supporting a link between decreased ARF expression and tumor development in patients with p53-wild type tumors. Thus, the novel MDM2-trap strategy, which addresses both the inhibition and mutations of p53, is an efficient strategy for the treatment of p53-mutated tumors. This article is protected by copyright. All rights reserved.

Dual Targeting of MDM4 and FTH1 by MMRi71 for Induced Protein Degradation and p53-Independent Apoptosis in Leukemia Cells.

MDM2 and MDM4 are cancer drug targets validated in multiple models for p53-based cancer therapies. The RING domains of MDM2 and non-p53-binder MDM2 splice isoforms form RING domain heterodimer polyubiquitin E3 ligases with MDM4, which regulate p53 stability in vivo and promote tumorigenesis independent of p53. Despite the importance of the MDM2 RING domain in p53 regulation and cancer development, small molecule inhibitors targeting the E3 ligase activity of MDM2-MDM4 are poorly explored. Here, we describe the synthesis and characterization of quinolinol derivatives for the identification of analogs that are capable of targeting the MDM2-MDM4 heterodimer E3 ligase and inducing apoptosis in cells. The structure-activity-relationship (SAR) study identified structural moieties critical for the inhibitory effects toward MDM2-MDM4 E3 ligase, the targeted degradation of MDM4 and FTH1 in cells, and anti-proliferation activity. Lead optimization led to the development of compound MMRi71 with improved activity. In addition to accumulating p53 proteins in wt-p53 bearing cancer cells as expected of any MDM2 inhibitors, MMRi71 effectively kills p53-null leukemia cells, an activity that conventional MDM2-p53 disrupting inhibitors lack. This study provides a prototype structure for developing MDM4/FTH1 dual-targeting inhibitors as potential cancer therapeutics.

Interferon-stimulated gene 15 enters posttranslational modifications of p53.

The tumor suppressor protein p53 is a central governor of various cellular signals. It is well accepted that ubiquitination as well as ubiquitin-like (UBL) modifications of p53 protein is critical in the control of its activity. Interferon-stimulated gene 15 (ISG15) is a well-known UBL protein with pleiotropic functions, serving both as a free intracellular molecule and as a modifier by conjugating to target proteins. Initially, attentions have historically focused on the antiviral effects of ISG15 pathway. Remarkably, a significant role in the processes of autophagy, DNA repair, and protein translation provided considerable insight into the new functions of ISG15 pathway. Despite the deterministic revelation of the relation between ISG15 and p53, the functional consequence of p53 ISGylation appears somewhat confused. More important, more recent studies have hinted p53 ubiquitination or other UBL modifications that might interconnect with its ISGylation. Here, we aim to summarize the current knowledge of p53 ISGylation and the differences in other significant modifications, which would be beneficial for the development of p53-based cancer therapy.

Dynamicsofp53:AMasterDeciderofCellFate.

Cellular stress-induced temporal alterations—i.e., dynamics—are typically exemplified by the dynamics of p53 that serve as a master to determine cell fate. p53 dynamics were initially identified as the variations of p53 protein levels. However, a growing number of studies have shown that p53 dynamics are also manifested in variations in the activity, spatial location, and posttranslational modifications of p53 proteins, as well as the interplay among all p53 dynamical features. These are essential in determining a specific outcome of cell fate. In this review, we discuss the importance of the multifaceted features of p53 dynamics and their roles in the cell fate decision process, as well as their potential applications in p53-based cancer therapy. The review provides new insights into p53 signaling pathways and their potentials in the development of new strategies in p53-based cancer therapy.

Induction Of p53 Transcription and Apoptosis By XPO1 Inhibition In Mantle Cell Lymphoma

We investigated the pathophysiological significance of MDM2 and the nuclear exporter Exportin 1 (XPO1/CRM1) on p53 transcription and their therapeutic targeting in mantle cell lymphoma (MCL). MCL is currently an incurable B-cell non-Hodgkin's lymphoma. Although TP53 mutations occur rarely (i.e., in 15 to 20% of the cases) in MCL, wild-type p53 is frequently inactivated by gene amplification of BMI1, homozygous deletion of CDKN2A (INK4a/ARF, over-expression of regulatory proteins, or gene deletion. For example, tumor cells may sequester p53 in the cytoplasm via the overexpression of proteins like MDM2 and XPO1 thereby abrogating its transcriptional anti-tumor activity. The selective MDM2 inhibitor Nutlin-3a (Vassilev, 2004) has been reported to induce p53-mediated apoptosis in MCL cells, and recently it was shown that the potent, small molecule Selective Inhibitor of Nuclear Export (SINE) induces apoptosis in MCL cells (Zhang, 2013).mRNA expression levels in MCL patient samples were determined using Oncomine (Compendia Bioscience, Ann Arbor, MI). MDM2 mRNA expression levels were not significantly higher in the MCL samples (n = 8) compared to normal B-cells ( n = 5) (P = 0.27). The MDM2 levels were also not associated with overall disease survival (P = 0.12) at 5 years (n = 63). In contrast, gene expression analysis on the same dataset demonstrated increased XPO1 mRNA expression in the MCL cells versus normal B-cells (P< 0.001). Furthermore, this higher XPO1 expression was associated with poorer prognosis in MCL patients (i.e., median overall survival: 3.2 years in low expression XPO1 cases and 1.9 years in high expression CRM1 cases, P = 0.033). mRNA levels of 11 representative p53 target genes were determined in 2 MCL (i.e., Z-138 and JVM-2) cells and were compared to those of four acute leukemia (i.e., OCI-AML3, MOLM-13, REH and NALM-6) cells. Cells from all six cell lines express wild-type p53. The MDM2 inhibitor Nutlin-3a (5 µM) poorly induced p53 target genes in the Z-138 (1.9 ± 0.12-fold) and JVM-2 cells (2.1 ± 0.23-fold) compared to acute leukemia cells (13.9 ± 5.2-fold) (P< 0.001 in both MCL cells), despite their increased p53 protein levels after Nutlin-3a treatment. The data suggest that p53-mediated transcription may be impaired in MCL via a Nutlin-induced increase in cytoplasmic p53 levels. Next, we treated Z-138 with 2 or 4 mM Nutlin-3a or with 80 or 160 nM KPT-185 for 18 hours and determined mRNA changes in p53 targets. KPT-185 induced p53 target genes more potently than Nutlin-3a (i.e., 2- to 4-times). To investigate the significance of p53-mediated apoptosis, Z-138 and JVM-2 were transduced with lentivirus encoding either negative control shRNA (ShC) or p53-specific shRNA (Shp53) and stable shRNA-expressing cells were generated. The p53-specific shRNA reduced p53 basal levels by 80 to 90%, and p53 knockdown cells were ∼80% less sensitive to Nutlin-3a and ∼60% to KPT-185, indicating that MDM2 or XPO1 inhibition rely on p53 pathway to induce apoptosis. Apoptotic induction by Nutlin-3a and KPT-185 in primary MCL cells were determined in 3 samples with wild-type p53. Treatment-specific Annexin V induction percentage in 2.5 µM Nutlin-3a-treated cells was 40.7 ± 8.0%, while it was 64.5 ± 15.0% in 100 nM KPT-185 treated cells. Treatment of 1 µM KPT-185 led to more prominent apoptosis, as evidenced by 81.8 ± 7.7% annexin V staining.Our data suggest that p53 is a component of MDM2 or XPO1 inhibition-induced apoptosis, and p53 transcription is more potently activated by XPO1 inhibition rather than MDM2 inhibition. Based on these data, we propose that active modulation of p53 by SINE XPO1 antagonism and/or MDM2 inhibition transcription would be beneficial in some cancer types to enhance p53-based cancer therapy. Disclosures:Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc.: Employment. Andreeff:Karyopharm Therapeutics Inc.: Research Funding.

A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX

The cellular response to p53 activation varies greatly in a stimulus- and cell type-specific manner. Dissecting the molecular mechanisms defining these cell fate choices will assist the development of effective p53-based cancer therapies and also illuminate fundamental processes by which gene networks control cellular behaviour. Using an experimental system wherein stimulus-specific p53 responses are elicited by non-genotoxic versus genotoxic agents, we discovered a novel mechanism that determines whether cells undergo proliferation arrest or cell death. Strikingly, we observe that key mediators of cell-cycle arrest (p21, 14-3-3σ) and apoptosis (PUMA, BAX) are equally activated regardless of outcome. In fact, arresting cells display strong translocation of PUMA and BAX to the mitochondria, yet fail to release cytochrome C or activate caspases. Surprisingly, the key differential events in apoptotic cells are p53-dependent activation of the DR4 death receptor pathway, caspase 8-mediated cleavage of BID, and BID-dependent activation of poised BAX at the mitochondria. These results reveal a previously unappreciated role for DR4 and the extrinsic apoptotic pathway in cell fate choice following p53 activation.

New Insights into p53 Signaling and Cancer Cell Response to DNA Damage: Implications for Cancer Therapy

Activation of the p53 signaling pathway by DNA-damaging agents was originally proposed to result either in cell cycle checkpoint activation to promote survival or in apoptotic cell death. This model provided the impetus for numerous studies focusing on the development of p53-based cancer therapies. According to recent evidence, however, most p53 wild-type human cell types respond to ionizing radiation by undergoing stress-induced premature senescence (SIPS) and not apoptosis. SIPS is a sustained growth-arrested state in which cells remain viable and secrete factors that may promote cancer growth and progression. The p21WAF1 (hereafter p21) protein has emerged as a key player in the p53 pathway. In addition to its well-studied role in cell cycle checkpoints, p21 regulates p53 and its upstream kinase (ATM), controls gene expression, suppresses apoptosis, and induces SIPS. Herein, we review these and related findings with human solid tumor-derived cell lines, report new data demonstrating dynamic behaviors of p53 and p21 in the DNA damage response, and examine the gain-of-function properties of cancer-associated p53 mutations. We point out obstacles in cancer-therapeutic strategies that are aimed at reactivating the wild-type p53 function and highlight some alternative approaches that target the apoptotic threshold in cancer cells with differing p53 status.

Inability of p53-reactivating compounds Nutlin-3 and RITA to overcome p53 resistance in tumor cells deficient in p53Ser46 phosphorylation

The p53 tumor suppressor protein plays key roles in protecting cells from tumorigenesis. Phosphorylation of p53 at Ser46 (p53Ser46) is considered to be a crucial modification regulating p53-mediated apoptosis. Because the activity of p53 is impaired in most human cancers, restoration of wild-type p53 (wt-p53) function by its gene transfer or by p53-reactivating small molecules has been extensively investigated. The p53-reactivating compounds Nutlin-3 and RITA activate p53 in the absence of genotoxic stress by antagonizing the action of its negative regulator Mdm2. Although controversial, Nutlin-3 was shown to induce p53-mediated apoptosis in a manner independent of p53 phosphorylation. Recently, RITA was shown to induce apoptosis by promoting p53Ser46 phosphorylation. Here we examined whether Nutlin-3 or RITA can overcome resistance to p53-mediated apoptosis in p53-resistant tumor cell lines lacking the ability to phosphorylate p53Ser46. We show that Nutlin-3 did not rescue the apoptotic defect of a Ser46 phosphorylation-defective p53 mutant in p53-sensitive tumor cells, and that RITA neither restored p53Ser46 phosphorylation nor induced apoptosis in p53Ser46 phosphorylation-deficient cells retaining wt-p53. Furthermore, treatment with Nutlin-3 or RITA together with adenoviral p53 gene transfer also failed to induce apoptosis in p53Ser46 phosphorylation-deficient cells either expressing or lacking wt-p53. These results indicate that neither Nutlin-3 nor RITA in able to induce p53-mediated apoptosis in the absence of p53Ser46 phosphorylation. Thus, the dysregulation of this phosphorylation in tumor cells may be a critical factor that limits the efficacy of these p53-based cancer therapies.

Coordination of the Nuclear and Cytoplasmic Activities of p53 in Response to DNA Damage

The tumor suppressor p53 plays a key role in the cellular response to various stresses. Most previous studies have focused on either the nuclear or cytoplasmic proapoptotic functions of p53, ignoring the combination of both functions. To explore how the two functions of p53 are coordinated in the DNA damage response via computer simulation, we construct a model for the p53 network comprising coupled positive and negative feedback loops involving p53, Mdm2, and Akt, as well as PUMA and Bax. In our model p53 is stabilized and accumulates in the nucleus and cytoplasm upon DNA damage. Nuclear p53 induces expression of Mdm2, PTEN, PUMA, and Bax. Cytoplasmic p53 is then released from the p53·Bcl-xL complex by PUMA to activate Bax directly. We find that the switching between low and high protein levels underlies the decision between cell survival and death. Moreover, a balance between the nuclear and cytoplasmic p53 levels and appropriate levels of Akt and PUMA are required for reliable cell fate decision. Our results indicate that coordination of the transcription-dependent and -independent activities of p53 is important in determining cellular outcomes. These findings advance our understanding of the mechanism for p53-mediated cellular responses and provide clues to p53-based cancer therapy.

P53-based Cancer Therapy

Inactivation of p53 functions is an almost universal feature of human cancer cells. This has spurred a tremendous effort to develop p53 based cancer therapies. Gene therapy using wild-type p53, delivered by adenovirus vectors, is now in widespread use in China. Other biologic approaches include the development of oncolytic viruses designed to replicate and kill only p53 defective cells and also the development of siRNA and antisense RNA's that activate p53 by inhibiting the function of the negative regulators Mdm2, MdmX, and HPV E6. The altered processing of p53 that occurs in tumor cells can elicit T-cell and B-cell responses to p53 that could be effective in eliminating cancer cells and p53 based vaccines are now in clinical trial. A number of small molecules that directly or indirectly activate the p53 response have also reached the clinic, of which the most advanced are the p53 mdm2 interaction inhibitors. Increased understanding of the p53 response is also allowing the development of powerful drug combinations that may increase the selectivity and safety of chemotherapy, by selective protection of normal cells and tissues.

P53-based cancer therapies: is defective p53 the Achilles heel of the tumor?

The tumor suppressor protein p53 plays a pivotal role in the DNA damage response and is defective in >50% of human tumors, which has generated substantial interest in developing p53-targeted cancer therapies. Various therapeutic rationales targeting p53 are currently under investigation including attempts to both activate and inhibit p53. Elevation of p53 can be achieved by either reintroducing an exogenous p53 gene or by blocking its association with its negative regulator hDM2. An alternate approach involves reverting mutant p53 to its wild-type conformation. Inhibition of p53 activity can be achieved either by preventing p53-mediated gene expression or by inhibiting the mitochondrial pro-apoptotic interactions of p53. These approaches are based on the concept that activation of p53 in a tumor is cytotoxic while inhibition of p53 in normal cells will protect the patient. However, activation of p53 also induces cell cycle arrest that can protect most normal cells from DNA damage, and this is the reason why many p53-defective tumors are more sensitive to DNA damage. The development of cell cycle checkpoint inhibitors to abrogate DNA damage-induced arrest builds on this observation as p53-defective cells appear particularly sensitive. Thus, normal cells are protected from premature entry into mitosis and the subsequent mitotic catastrophe induced by checkpoint inhibitors, while p53-defective tumor cells are destroyed. These contradictory approaches must be resolved if we are to take full advantage of the frequent p53 defect in tumors.

Prospects for p53-based cancer therapy.

The p53 tumor suppressor plays the role of a cellular hub which gathers stress signals such as damage to DNA or hypoxia and translates them into a complex response. p53 exerts its action mainly as a potent transcription factor. The two major outcomes of p53 activity are highlighted: cell cycle arrest and apoptosis. During malignant transformation p53 or p53-pathway related molecules are disabled extremely often. Mutations in p53 gene are present in every second human tumor. A mutant form of p53 may not only negate the wild type p53 function but may play additional role in tumor progression. Therefore p53 represents a relatively unique and specific target for anticancer drug design. Current approaches include several different molecules able to restore p53 wild-type conformation and activity. Such small molecule drugs hold great promise in treating human tumors with dysfunction of p53 pathway in the near future.

Novel human p53 mutations that are toxic to yeast can enhance transactivation of specific promoters and reactivate tumor p53 mutants.

Since highly expressed human p53 can inhibit human and yeast cell growth, we predicted that p53 mutants could be generated with increased growth inhibition of the yeast Saccharomyces cerevisiae and that these would be useful for characterizing p53 functions and tumor p53 mutants. A random mutagenesis screen led to the isolation of mutations in the DNA binding domain that result in p53 being lethal even at moderate expression levels in yeast. Three independent mutants had an alanine change at the evolutionary invariant V122 in the L1 loop. The other toxic mutations affected codons 277 (C277R, C277W) and 279 (G279R). This latter amino acid change was also reported in tumors, while all the other mutations are novel. A recently developed rheostatable GALI promoter system that provides graded increases in expression of p53 was used to examine the transactivation function of the toxic mutations when expression was greatly reduced and cells were viable. At low expression levels the toxic mutants lacked transactivation from a 3xRGC responsive element (RE). Surprisingly some exhibited enhanced transactivation with p21 and bax REs. The V122A mutant was able to re-activate transactivation of various p53 tumor mutants and retained growth inhibition when co-expressed with dominant-negative tumor mutations. Upon expression in human Saos-2 cells the V122A p53 mutant caused growth suppression, was capable of transactivation and exhibited higher than wild type activity with the bax promoter in luciferase assays. A non-functional p53 tumor mutant was partially reactivated by V122A for both transactivation and growth suppression. Thus, the screen for toxic p53 mutants in yeast can identify novel p53 variants that may be useful in dissecting p53 regulated cellular responses and in developing p53-based cancer therapies.

Precise characterisation of monoclonal antibodies to the C-terminal region of p53 protein using the PEPSCAN ELISA technique and a new non-radioactive gel shift assay

The development of human cancers is frequently associated with inactivation of the p53 tumour suppressor protein triggering cell cycle arrest or apoptosis in response to cellular stress. The p53 protein has been identified as a transcription factor with sequence-specific DNA binding properties. The DNA-binding activity is cryptic but can be modulated through the C-terminal region of the p53 protein by several different stimuli, including phosphorylation by casein kinase II (CKII), protein kinase C (PKC) or binding of the C-terminal monoclonal antibody PAb421. Monoclonal antibodies to the C-terminal region of p53 protein are able to activate the latent form of p53 and induce binding to DNA. To characterise such antibodies, we used a combination of the PEPSCAN ELISA procedure and a newly developed non-radioactive gel shift assay. Monoclonal antibodies from the Bp53 series displayed higher affinities for the human, rat and mouse p53 proteins than did the conventional antibody PAb421. In addition, these antibodies were able to activate the sequence-specific DNA binding functions in latent forms of p53 protein and, in contrast to PAb421, they were able to recognise both PKC phosphorylated and PKC non-phosphorylated forms of p53 protein. Our monoclonal antibodies recognising post-translationally modified target epitopes in the C-terminal region of p53 protein might assist the development of more effective molecules for p53-based cancer therapy.

Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellular expression of anti-p53 single chain Fv fragments.

We report here the production and the properties of single chain Fv fragments (scFvs) derived from the anti-p53 monoclonal antibodies PAb421 and 11D3. 11D3 is a newly generated monoclonal antibody which exhibits properties very comparable to those of PAb421. The scFvs PAb421 and 11D3 are able to stably associate with p53 and to restore the DNA binding activity of some p53 mutants in vitro. When expressed in p53 -/-human tumour cells, the scFv421 is essentially localized in the cytoplasm in the absence of p53, and in the nucleus when exogenous p53 is present. Thus, p53 is also able to stably associate with an anti-p53 scFv in cells. Cotransfection of p53 -/- human tumour cells with expression vectors encoding the His273 p53 mutant and either scFv leads to restoration of the p53 mutant deficient transcriptional activity. These data demonstrate that, in human tumour cells, these scFvs are able to restore a function essential for the tumour suppressor activity of p53 and may represent a novel class of molecules for p53-based cancer therapy.